This invention is directed towards synthetic ribonucleic acid (RNA), analogs, and aptamers which have been capped in vitro and to methods of making them.
Most viral and cellular mRNA molecules contain a 5xe2x80x2-methylated cap structure. The presence of such a structure is important for mRNA maturation, initiation of translation and protects the mRNA against degradation by various RNases present in the cell. There are various types of RNA caps known. The general structure of a capped RNA can be designated as m7G(5xe2x80x2)ppp(5xe2x80x2)Pu, (where Pu, the penultimate base, is typically a purine nucleoside).
In the so-called xe2x80x9cCap 0xe2x80x9d, the penultimate base is unmodified. xe2x80x9cCap 0xe2x80x9d is found commonly in yeast, the majority of slime molds, and in plant viruses.
The penultimate base of xe2x80x9cCap 1xe2x80x9d containing mRNAs is 2xe2x80x2-O-methylated and can be designated m7G(5xe2x80x2)ppp(5xe2x80x2)Pu(2xe2x80x2-OMe). It is formed as a result of the action of a 2xe2x80x2-O-methyltransferase activity. Many mRNAs from animal viruses have a xe2x80x9cCap 1xe2x80x9d structure. The presence of the 2xe2x80x2-O-methyl group stabilizes the first 3xe2x80x2,5xe2x80x2-phosphodiester linkage of xe2x80x9cCap 1xe2x80x9d-containing mRNAs against RNase T2 cleavage.
Messenger RNAs having a xe2x80x9cCap 1xe2x80x9d structure have two 2xe2x80x2-O-methyl groups: one on the penultimate base and the second on the next base 3xe2x80x2 to the penultimate base [m7G(5xe2x80x2)ppp(5xe2x80x2)Pu(2xe2x80x2OMe)xc3x97(2xe2x80x2OMe)]. This is found in silk fibroin mRNA, vesicular stomatitis virus mRNA and other cellular and viral mRNAs. The presence of caps and their type can be readily determined by those skilled in the art; one method includes treating the mRNA with T2 RNase and alkaline phosphatase and analyzing the digest by DEAE-cellulose chromatography.
Influenza virus endonuclease uses a capped RNA as its substrate. Detailed enzymological studies of this endonuclease have been hindered in the past because it is quite difficult to synthesize capped RNAs of desired purity and/or capped RNAs which contain analogs. Further, in view of the synthesis problems, it has been impossible to identify well defined short capped RNA or RNA analog molecules which could be used as substrates or inhibitors of influenza endonuclease, and which could be potential therapeutic and/or prophylactic agents.
Aptamers are single-stranded or double-stranded nucleic acids which are capable of binding proteins or other small molecules. Aptamers which have therapuetic value would most likely bind proteins involved in the regulation and expression of genes, such as transcription factors. The presence of the aptamer would act as a sink for the protein A factors, preventing the factors from carrying out their normal functions and presumably modulating the expression of genes dependent upon the activity of this protein. To date, only a few aptamers are known. It would be desirable to identify novel aptamers active against enzymes such as influenza endonuclease and to be able to easily sythesize them.
In the past, capped RNA molecules have been made in vitro by xe2x80x9crunoffxe2x80x9d transcription. There are two routes to obtaining a capped RNA by this procedure. The first is to carry out an RNA synthesis reaction using a DNA template and an RNA polymerase, such as T7 RNA polymerase, in the presence of a capped dinucleotide such as m7G(5xe2x80x2)ppp(5xe2x80x2)G. A second methodology carries out the runoff transcription reaction and the resulting RNA is then treated with an enzyme, guanylyltransferase, which xe2x80x9ccapsxe2x80x9d the RNA.
These two methods suffer from several problems. First, while both methods are generally efficient for producing long polyribonucleic acids (i.e., more than approximately 100 bases), they do not generally work well for shorter nucleotides (i.e., less than about 40 bases). There are also problems with maintaining fidelity of the sequence of the resulting oligoribonucleotide, as transcriptional errors cannot be ruled out during xe2x80x9crunoffxe2x80x9d transcription. The major disadvantage of the first method is that a low percentage of the RNAs produced will contain the Cap. Further, the RNA product will have a Cap 0 structure, and certain enzymes such as influenza endonuclease requires a Cap 1 structure to bind. Secondly, 2xe2x80x2-O-methylation is generally inefficient and the extent of methylation cannot be ascertained in a simple manner.
Another major limitation of this methodology is that the vast majority of nucleoside (or non-nucleoside) analogs cannot be incorporated into the RNA in a site specific manner. Furthermore, there is difficulty in synthesizing RNAs containing such analogs because the analogs are not utilized as substrates by the polymerase. Similarly, in the case of RNA analogs which contain backbone modifications such as phosphorothioates or methylphosphonates, usually only one of the corresponding diastereoisomers is recognized as a substrate by the polymerase.
It would be desirable to have a simplified, efficient method for producing short capped unmodified or modified RNAs.
This invention is directed to a method for producing capped ribonucleic acid molecules, analogs, and aptamers comprising the steps of: a) reacting an RNA or RNA analog oligonucleotide with a phosphate addition agent to form an RNA or RNA analog mono-, di- or triphosphate; and b) capping the RNA or RNA analog mono-, di- or triphosphate.
This invention specifically is directed to a method of producing capped RNA oligonucleotides, capped RNA analog oligonucleotides, and aptamers comprising the steps of: a) reacting an RNA oligoribonucleotide or RNA analog oligonucleotide with a phosphate addition agent to form a first intermediate; b) reacting the first intermediate with a phosphate analog to form a cyclic triphosphate intermediate or a diphosphate intermediate; c) oxidizing and hydrolyzing the cyclic triphosphate intermediate or diphosphate intermediate to result in a triphosphate or diphosphate; and d) capping the diphosphate or triphosphate to form a capped RNA, capped RNA analog, or aptamer.
This invention further relates to particular capped RNAs and capped RNA analogs which may be produced by the process of this invention. These molecules are substrates for influenza endonuclease, and as such can be used to gain new insights as to the mechanism of the endonucleases. This invention further relates to specific aptamers which are substrate inhibitors of influenza endonuclease, an enzyme which is critical to the replication and resulting infectivity of the influenza virus. Thus, another aspect of this invention is a method of preventing or treating influenza in an animal, including humans, which is susceptible to infection by the influenza virus by administering an effective amount of an influenza endonuclease aptamer.